1. Technical Field
The invention relates, in general, to a low molecular weight mimic of superoxide dismutase and, in particular, to a desferrioxamine-manganese complex capable of scavenging superoxide radicals.
2. Background Information
The superoxide radical (O.sub.2.sup.-) can be generated within living cells during both enzymic and non-enzymic oxidations. Because of the direct reactivity of O.sub.2.sup.-, and the reactivity of secondary free radicals that it can generate, O.sub.2.sup.- presents a threat to cellular integrity. This threat is met by a family of defensive enzymes that catalyze the conversion of O.sub.2.sup.- to H.sub.2 O.sub.2 +O.sub.2. These enzymes, superoxide dismutases (SOD), react with O.sub.2.sup.- at a rate that approaches the theoretical diffusion limit and appear to be important for aerobic life. The H.sub.2 O.sub.2 generated by SOD is disposed of either by catalytic conversion to O.sub.2 and H.sub.2 O by catalases, or by reduction to water at the expense of thiol, amine or phenolic substrates by peroxidases.
The superoxide radical has been shown to be an important causative factor in the damage resulting from: a) autoxidation; b) oxygen toxicity; c) the oxygen-dependent toxicity of numerous compounds; d) reperfusion injury; e) inflammation; and f) frostbite; and is implicated in the limited viability of transplanted organs and tissues.
The earliest work bearing on the functions of SOD dealt primarily with oxygen toxicity and with the oxygen-dependent toxicities of viologens, quinones and related redox-cycling compounds. These investigations established that O.sub.2.sup.-, made within cells, can kill the cells and that SOD provides a defense It is now known that O.sub.x.sup.- is not only an unwanted and dangerous byproduct of dioxygen metabolism, but is also produced in large quantities by certain specialized cells, seemingly to serve a specific purpose. Neutrophils, and related phagocytic leucocytes, contain a membrane-associated NADPH oxidase that is activated when the cells are stimulated and that specifically reduces dioxygen to O.sub.2.sup.31 . A defect in this enzyme weakens the microbicidal activity of these leucocytes, leading to chronic granulomatous disease.
The known association of neutrophils with the inflammatory process, and the production of O.sub.2.sup.- by activated neutrophils, suggests a role for O.sub.2.sup.- in the development, and possibly in the deleterious consequences, of inflammation. An enzymic source of O.sub.2.sup.- decreases the viscosity of synovial fluid by depolymerizing hyaluronate and SOD exerts a protective effect. Injecting an enzymic source of O.sub.2.sup.-, such as xanthine oxidase, causes a localized inflammation that can be prevented by scavengers of oxygen radicals, such as SOD.
The anti-inflammatory effect of SOD, noted in model inflammations in laboratory animals, is explained in terms of the inhibition of the production of a neutrophil chemotaxin by the reaction of O.sub.2.sup.- with a precursor present in normal human serum. SOD, when injected into the circulation, is rapidly removed by the kidneys, such that the circulation half life of i.v.-injected bovine SOD in the rat is only 7 minutes. This can be markedly increased by coupling the SOD to polyethylene glycol or ficoll, with a corresponding increase in anti-inflammatory effect.
The tissue damage that develops as a consequence of temporary ischemia has classically been attributed to the lack of ATP which develops during the hypoxia imposed during ischemia. Data support the view that this damage actually occurs during reperfusion and is an expression of increased oxygen radical production. SOD protects against this reperfusion injury.
The mechanism which best fits these data depends upon degradation of ATP to hypoxanthine and upon the conversion of xanthine dehydrogenase to xanthine oxidase, during the period of ischemia. Reperfusion then introduces dioxygen, which is reduced to O.sub.2.sup.- by the action of xanthine oxidase on the accumulated hypoxanthine. As expected from this model, allopurinol, which inactivates xanthine oxidase, also protects against reperfusion injury.
The superoxide dismutases are used as pharmacological agents. They are applied to the treatment of inflammatory diseases and are being investigated in the cases of the reperfusion injury associated with skin grafts, organ transplants, frostbite and myocardial infarction. Size, antigenicity and cost, however, mitigate against their widespread usage. Since the enzyme must be isolated from biological sources, it is in limited supply, very expensive and plagued by problems caused by contaminants.
It has long been apparent that low molecular weight mimics of SOD, capable of acting intracellularly, would be useful. Manganese(II), per se, will scavenge O.sub.2.sup.- and, in suitable buffers, will do so catalytically. However, Mn(II) binds avidly to a number of proteins and in so doing loses its activity. Cu(II) is itself a very effective catalyst of the dismutation of O.sub.2.sup.-. Since the first SOD to be discovered was a copper protein, copper-complexes have been examined for SOD activity. The problems with free Cu(II) are that it readily forms a hydroxide and that it binds strongly to many macromolecules. For these reasons Cu(II) per se is most active in acid solutions and in the absence of strongly binding ligands. Among the complexes of Cu(II), the SOD-like activity for which have been reported, are: Cu(lys)2 and Cu(gly-his).sub.2, Cu(diisopropylsalicylate).sub.2, Cu(penicillamine), Cu(histidine), Cu(dipeptides) and Cu(gly-his-lys). There are serious problems with all of these copper complexes. Many are, in fact, merely acting as metal buffers, serving to solubilize the Cu(II) and are of insufficient stability to retain activity in the presence of serum albumin. Investigations of Cu(II) complexes have thus far not resulted in the discovery of any biologically useful mimics of SOD.